CRISPR/Cas systems have recently been described to mediate bacterial defense against invading foreign nucleic acid derived from bacteriophages or plasmids, which they target for degradation (1, 2). These systems have not previously been shown to target mRNA or control endogenous gene expression. We demonstrated that the CRISPR/Cas protein Cas9 targets an endogenous mRNA, revealing a novel bacterial RNA silencing machinery and genetic regulatory paradigm (3). Cas9 functions with 2 small RNAs to repress an endogenous bacterial lipoprotein (BLP) in the intracellular pathogen Francisella novicida (3). Since BLPs trigger a proinflammatory innate immune response through Toll-like Receptor 2 (TLR2)(4, 5), aimed at combating pathogens, CRISPR/Cas-mediated repression of BLP is critical for F. novicida to dampen this host response and is critical for virulence in vivo 3, 6). Interestingly, our data show that the Cas9 regulatory system is activated when the bacteria are in the macrophage phagosome, which contains TLR2 (3, 6). There, the bacteria are confronted with numerous antimicrobials that induce membrane damage (7), which we hypothesize is the trigger for the induction of the Cas9 system, leading to evasion of TLR2. Cas9 is encoded by at least 63 bacterial pathogens (as well as at least 22 commensals)(3, 8), and we have demonstrated its role in virulence traits of Neisseria meningitidis (3), while others have recently shown a role in Campylobacter jejuni (9). These data suggest that the Cas9 system is broadly used to mediate the interaction of bacterial pathogens, as well as numerous commensals, with eukaryotic hosts. Since Cas9 targeting of a BLP mRNA in F. novicida is the only currently known example of CRISPR/Cas- mediated endogenous gene regulation, we will employ this model to answer fundamental questions about the mechanism of action of this system, as well as the parameters controlling its induction. This will lead to the elucidation of basic foundational principles governing Cas9 and CRISPR/Cas biology. We will study the Cas9 regulatory system in N. meningitidis, using the insights we gain from experiments with F. novicida as a guide, likely serving to both strengthen our knowledge of core components present in both systems, as well as identifying potential differences. The proposed research will have a sustained and powerful impact on our understanding of Cas9, CRISPR/Cas systems, RNA silencing, genetic regulatory mechanisms, bacterial virulence, and innate immune evasion, and lay the framework for a much broader knowledge of how diverse bacterial pathogens cause disease.